Tissue engineering, transplantation therapy, gene therapy, and drug discovery promise to revolutionize the medical paradigm and herald the coming of the “biotechnology era”. However, development and implementation of these technologies depend upon cell-based approaches that require a plentiful supply of human cells: a supply that unfortunately does not currently exist. Therefore, identifying sources of human cells that can be practically obtained in sufficient quantities is of paramount importance. Spheroid forming cells, preferably pluripotent cells such as human embryonic stem cells (Thomson, Itskovitz-Eldor et al. 1998), neural stem cells (Clarke, Johansson, et al. 2000), multipotent adult progenitor cells (Schwartz, Reyes, et al. 2002), and others may be able to provide tissue in this manner.
The potential utility of pluripotent cells, in particular, the utility of embryonic stem (ES) cells, is derived from their demonstrated ability to differentiate into any other cell type in the body (Evans and Kaufman, 1981). Derived from the early embryo, ES cells can be grown and manipulated in vitro. ES cells are pluripotent cells that have the ability to differentiate into any cell type in the body (Evans and Kaufman 1981). They also have the ability to grow and multiply indefinitely while maintaining their pluripotentiality. Theoretically, these traits make ES cells an unlimited source of any cell type and therefore a very attractive tissue source for other biotechnological applications (Zandstra and Nagy 2001).
Generation of various differentiated cell types (cardiac myocytes, hematopoietic cells, neurons, hepatocytes, and others) from murine ES cells (e.g., Wang, Clark et al. 1992; Rohwedel, Maltsev et al. 1994; Bain, Kitchens et al. 1995; Palacios, Golunski et al. 1995; Choi, Kennedy et al. 1998; Fleischmann, Bloch et al. 1998; Tropepe, Sibilia et al. 1999) as well as from human ES cells (e.g., Jones and Thomson 2000; Thomson et al. 1998; Mummery et al 2002; Levenberg et al. 2002; Reubinoff et al. 2001; Assady et al 2001) has been extensively reported.
In general, production of these various cell types from ES cells require a complex multi-step process, starting with the expansion of ES cells to adequate numbers. Next, ES cells would be differentiated to the desired cell type(s). Finally, the desired cells would be selected and purified from the remaining cells before they could be utilized for various applications.
In vitro, differentiating ES cells follow a reproducible temporal pattern of development that in many ways recapitulates early embryogenesis (Keller 1995). As ES cells and their derivatives proliferate and differentiate, they typically form spherical tissue-like structures (spheroids) called Embryoid Bodies (EBs). Other pluripotent stem cells such as neural stem cells have also been described to form spheroids. In the case of neural stem cells these spheroids are termed neurospheres (Kalyani, Hobson, and Rao, 1997). In the case of ES cells, over time, EBs increase in cell number and complexity as cells from the three embryonic germ layers are formed (Keller 1995). The ability to control and manipulate the formation of spheroids from pluripotent cells and other types of cells that are derived directly, or indirectly from human tissue, as well as the interactions between the spheroids and between the spheroid-forming cells (i.e., prevent or inhibit the aggregation of spheroids) would allow for the control of interactions between these cells during proliferation and differentiation, and thus be an important development for the design of technologies for the in vitro differentiation of pluripotent cells. For example, the generation of “chimeric spheroids” or “chimeric EBs” defined as multicellular, spherical structure comprising a mixture of differentiated cells and pluripotent cells (Perkins, 1998), has been shown to be an effective approach to influence the development of the cell types in from pluripotent cells (Clarke, Johansson et al. 2000).
Current ES cell differentiation systems include cultures initiated with an ES cell suspension in liquid media (LSC), or methylcellulose (semi-solid) media (MC), or attached to a surface in liquid media, or with multi-ES cell aggregates formed in “hanging drop” cultures (HD) where ES cells are aggregated in hanging drops for 2 days before transfer to liquid culture (see FIG. 1). These systems are adequate for small-scale laboratory purposes but are not amenable to clinical production because of deficiency in three important areas: a) the ability to measure and control the extracellular environment, b) scalability, and c) cell density.
These systems are static and batch-style cultures that result in formation of spatial and temporal gradients in nutrients and metabolic products. These gradients make measurement and process control difficult because these processes depend on sampling measurements that need to reflect the conditions throughout the culture. The inability to control the cell culture enviroment will affect product purity and reproducibility, as well as the cell types that can be readily generated [e.g.; for oxygen (Maltepe, Schmidt, et al. 1997), glucose (Soira, 2001) and many other biological and physicochemical factors (Zandstra and Nagy, 2001)]. In the case of methylcellulose culture, the semi-solid media also hinders measurement and manipulation. Regulatory approval for the use of any biotechnology product demands consistency in the method of production and in the final product itself (http://www.fda.gov/cdrh/tisseng/te6.html). This is not possible when measurement and control are impeded or absent.
Second, clinical or industrial scale production of cells from the current culture systems is not practical. The current systems are typically carried out in petri dishes where cells grow in only a thin layer of media. These systems are essentially two-dimensional with respect to the EBs; therefore, unrealistically large surface areas would be required for industrial scale production. It can be estimated that several billion cells would be need for applications such as cardiac cell therapy (Zandstra and Nagy, 2001); this scale of cell production is highly impractical, if not impossible using current technologies.
Third, current liquid suspension cultures (LSC) require EBs to be cultured at relatively low density. Higher cell densities can be achieved in methylcellulose cultures, where semi-solid media reduces the likelihood of cell aggregation, however media supplementation of methylcellulose cultures is troublesome and increased cell density would demand more frequent supplementation.
Because of the limitations reviewed above, one ideal differentiation culture system for clinical scale production of EBs would be the stirred tank reactor or other scalable, well-mixed and controlled bioreactor systems such as a fluidized bed reactor. While the standard methods are limited to two-dimensional growth because of static conditions, a controlled bioreactor could suspend the cells evenly throughout the volume (in three-dimensions). This configuration of system can be easily scaled in size to accommodate the need for increased production of cells. In addition, stirring ensures homogenous media conditions throughout the reactor that would facilitate measurement and control of the extracellular environment.
Despite the importance of stirred or other well-mixed bioreactors, to date there have been no reports of successful use of these systems for ES cell differentiation culture. ES cells added directly to stirred culture quickly aggregate into large cell clumps within 24 hours and cell growth and differentiation within these clumps are severely impaired (Wartenberg et al. (1998)). Differentiation of ES cells in stirred culture is therefore a nontrivial task; however the ability to do so would greatly facilitate clinical and industrial scale production of cells for therapeutic purposes. ES cells cultured in stirred bioreactors fail to generate EBs in an efficient manner. Therefore, there is a need to develop methods for efficient growth and differentiation of spheroid forming cells, preferably pluripotent cells, such as ES cells.